This invention relates to a method of improving protein production, particularly antibody production, in animal cell cultures. Briefly, hypertonic rather than isotonic media are used to feed and maintain the cells. This has the effect of increasing cell viability. Such hypertonic media are especially well suited for growing antibody-producing cells such as hybridoma since such cells produce much more antibody without significant modification of their viability or growth rate.
Conventional media for cultivation of mammalian cells are formulated in accordance with Eagle's two seminal papers on the effect of ion concentration on cell growth cycles, see Science, 122: 501-4(1955) and Arch. Biochem. & Biophys., 61;356-66 (1956). Eagle determined that media containing 85-115 mM sodium and 1-10 mM potassium were optimum for growth of both Hela cells and mouse fibroblasts. The formulation of Eagle's basal medium, which forms the basis for almost all animal cell growth media used today, is based on these results.
Eagle's work was followed by Stubblefield and Mueller, see Cancer Res. 20: 1646-1655 (1960), who explored the effects of sodium chloride concentration on growth, biochemical composition, and metabolism of HeLa cells. Stubblefield discovered that higher concentrations of sodium chloride in the growth medium prevented cell replication, leading to a decrease in the number of cells, while promoting RNA and protein synthesis in the cells that survive. Stubblefield theorized that the change in saline content hindered DNA replication thereby preventing mitosis and permitting the cells which survived to grow larger than they otherwise would. The addition of sodium chloride changed the osmolarity of the medium but Stubblefield did not attempt to differentiate between osmotic and ionic effects.
Twelve years later, Schachtschabel and Foley (see Exp. Cell Res. 70: 317-324, 1972) cultured Ehrlich ascites tumor cells in a media made hypertonic by the addition of sodium chloride to determine the effect of high tonicity media on the growth cycle of this type of cells. Schachtschabel found that a salt tolerant population which followed a substantially normal life cycle developed after significant losses in the number of cells in the culture. However, these salt-tolerant cells had a somewhat different morphology than conventional cells, i.e., they appeared more like fibroblast than epithelial cells. The generation or doubling time for these cells increased dramatically and the number of surviving cells decreased as the tonicity of the media was increased.
Antibody-producing cultures, e.g., spleen cells and hybridomas, are normally grown in media such as Eagle's modified media containing serum. Growth cycles for the antibody-producing cells in these media and the amount of antibody secreted is within the expected range as compared with other protein-secreting cells grown in these media. However, the increased use of antibodies as biological tools, particularly through the advances in hybridoma and monoclonal antibody formation techniques, have led to a need for increased antibody production from specific cell cultures.
Myelomas are also grown normally in isotonic media. Since these cells produce protein and can be genetically modified, a medium which could increase protein production level without loss of cell viability would be advantageous.
Accordingly, an object of the invention is to provide a method of culturing protein-producing cells to improve protein production. Another object is to promote high antibody production from antibody-producing cells while developing a viable, expanding culture. A further object is to promote hybridoma growth and monoclonal antibody production without adding feeder cells to the culture. A still further object of the invention is to provide a method of obtaining high protein yield from large volume cultures containing a high density of cells. Another object of the invention is to promote protein production from cells, e.g., myeloma, while retaining cell viability. These and other objects and features of the invention will be apparent from the summary, the drawing, and the description which follow.